Deuterated piperazine compound as well as preparation method and application thereof

Abstract

The invention provides a deuterated piperazine compound as well as a preparation method and application thereof. The compound has a structure as shown in a formula I. The invention further relates to a preparation method of the compound with the structure shown in the formula I, a pharmaceutical composition and application of the compound in preparation of anti-SARS-CoV-2Mpro drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a derivative and a preparation method thereof, and in particular to a deuterated piperazine compound, a composition comprising the compound, the preparation of the compound and application of the compound in the field of anti-coronavirus drugs, belonging to the technical field of organic synthesis and pharmaceutical application. Background Art

[0002] SARS-CoV-2 seriously threatens human life and health, affects social and economic development, and the new coronavirus infection is not over. SARS-CoV-2 mutates rapidly and has a significant ability to escape immunity. It can break through the immune barrier formed by vaccines or infections in the human body and cause multiple infections. The new coronavirus is likely to coexist with humans for a long time like the influenza virus. Although there are many drugs on the market, the new coronavirus is very easy to mutate and quickly develop drug resistance; the continuous mutation of SARS-CoV-2 and the interspecies similarity of coronaviruses highlight the urgent need to design and develop broad-spectrum anti-resistant coronavirus therapeutics. It is necessary to develop anti-coronavirus drugs for targets with broad-spectrum and conservative properties.

[0003] SARS-CoV-2 main protease (M pro ) is a homodimeric cysteine ​​protease composed of two mutually perpendicular monomers, and its catalytic center is composed of Cys145 and His41 (see Figure 1 The main protease plays an important role in the viral replication cycle and is a common protease with high similarity among coronavirus species, among which SARS-CoV-2 and SARS-CoVM pro With 96% sequence homology. pro The high conservation of sequence and structure makes it an ideal target for the development of anti-coronavirus drugs. pro Inhibitors have been reported successively, mainly including peptidomimetic compounds, such as the currently marketed peptidomimetic M pro Covalent inhibitors: Namatevir, Senotevir and Leretevir; non-peptide non-covalent inhibitors Ensetevir and GC-78 (discovered by the applicant), etc. In addition, FB2001, which is currently in the clinical stage, is also a peptidomimetic covalent inhibitor, but their structure is single and highly homogenized. Moreover, due to the high mutability of the new coronavirus and the increase in drug use, SARS-CoV-2 rapidly develops drug resistance. Currently, Namatevir-resistant strains (E166V / T21I and E166V / L50F double mutants) have appeared in the clinic. Therefore, it is urgent to discover anti-coronavirus drugs with unique skeletons and broad-spectrum resistance, and also to prepare drug reserves and technical support for possible future outbreaks.

[0004]

[0005] Dichlorophenylpiperazine compounds GC-78 (IC 50 =0.46 μM, EC 50 =0.89μM) is a new type of SARS-CoV-2 non-peptide main protease inhibitor with a new skeleton. However, its activity is still lower than that of the marketed drug Nematevir, and its pharmacokinetic properties are poor (t 1 / 2 =1.64h; F<20%), further optimization and improvement is needed. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a deuterated piperazine compound, a composition comprising the compound and a preparation method thereof, and the present invention also provides the above-mentioned compound as a SARS-CoV-2 M pro Activity screening results of inhibitors and their applications.

[0007] The technical solution of the present invention is as follows:

[0008] 1. Deuterated piperazine compounds

[0009] The deuterated piperazine compound, or a pharmaceutically acceptable salt thereof, has a structure shown in the following general formula I:

[0010]

[0011] in,

[0012] R1 and R2 are: methoxy, deuterated methoxy or methylthio, each independently;

[0013] The compound represented by formula I is in S configuration.

[0014] Pharmaceutically acceptable salt forms include commonly used inorganic acid salts such as hydrochloride, sulfate or phosphate, and organic acid salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate or acetate.

[0015] Preferably according to the present invention, the deuterated piperazine compound is one of the following compounds:

[0016]

[0017] The "pharmaceutically acceptable salt" described in the present invention refers to a salt of a compound that is suitable for contact with human or lower animal tissues without undue toxicity, irritation, and allergic reactions within a reliable pharmaceutical evaluation range, has a fairly reasonable benefit-risk ratio, is usually water- or oil-soluble or dispersible, and can be effectively used for its intended use. It includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which can be used for the intended purpose and are compatible with the chemical properties of the compounds of formula I and II. A list of suitable salts is shown in SM Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19. Further preferred salt types are inorganic acid salts such as hydrochlorides, sulfates or phosphates, and organic acid salts such as methanesulfonates, p-toluenesulfonates, fumarates, citrates or acetates.

[0018] 2. Preparation method of deuterated piperazine compounds

[0019] The preparation method of deuterated piperazine compounds comprises the following steps: using 3,4-dichlorophenylboronic acid 1 and (S)-Boc-piperazine carboxylate 2 as starting materials and dichloromethane as a reaction solvent, an intermediate 3 is obtained by a Chan-Lam coupling reaction under the catalysis of copper acetate; the methyl ester is then hydrolyzed by lithium hydroxide to obtain an intermediate 4; the intermediate 4 is subsequently dissolved in an appropriate amount of dichloromethane, and condensed with various R1s under the action of a condensation agent HATU to obtain an intermediate 5; the intermediate 5 is then deprotected by trifluoroacetic acid to obtain an intermediate 6; and the intermediate 6 is then subjected to an amide condensation reaction with various substituted nicotinic acids in dichloromethane under the action of HATU to obtain various target products;

[0020] The synthetic route is as follows:

[0021]

[0022] Reagents and conditions: (i) acetone, oxygen, pyridine, dichloromethane, room temperature; (ii) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (iii) deuterated p-methoxyaniline or p-methoxyaniline, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; (iv) 2M HCl-dioxane, dichloromethane, room temperature; (v) deuterated methoxynicotinic acid or methylthionicotinic acid, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature.

[0023] Wherein, R1 and R2 are: methoxy, deuterated methoxy and methylthio, each independently;

[0024] The room temperature described in the present invention is 20-30°C.

[0025] According to a preferred method of the present invention, the preparation method of the deuterated piperazine compound comprises the following specific steps:

[0026] (1) Add (S)-1-tert-butyl-3-methylpiperazine-1,3-dicarboxylic acid (1) and 3,4-dichlorophenylboronic acid (2) to dichloromethane and dissolve until almost clear during stirring; add anhydrous copper acetate and 2 equivalents of pyridine to the solution at once; react for 24 hours under an oxygen atmosphere, and detect the reaction by TLC: ethyl acetate / petroleum ether = 1:2, v / v; after the reaction is complete, add water to quench the reaction; separate the liquids, and repeatedly wash with distilled water until the organic phase has no blue color; wash the organic phase with a saturated sodium chloride solution, separate the organic phase, and successively dry it over anhydrous sodium sulfate, filter it, and concentrate it under reduced pressure. The resulting crude product is separated and purified by silica gel column chromatography to obtain intermediate 3, which is a colorless oily liquid;

[0027] (2) Intermediate 3 was dissolved in a mixture of methanol and tetrahydrofuran, and an aqueous solution of lithium hydroxide was added dropwise under ice bath, reacted at room temperature, stirred for 4 hours, and then concentrated under reduced pressure to remove the organic solvent; 1M dilute hydrochloric acid was added dropwise to the remaining aqueous solution under ice bath to adjust the pH to below 4, and a large amount of milky white solid precipitated. Intermediate 4 was obtained by filtration, washing, and drying as a white powdery solid;

[0028] (3) Under ice bath, intermediate 4 and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were added to dichloromethane, activated for 30 min, and then N,N'-diisopropylethylamine and deuterated p-methoxyaniline or p-methoxyaniline were added. The mixture was allowed to react at room temperature for 20 h. The reaction was monitored by TLC until completion. Water was added to quench the reaction. The dichloromethane phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain intermediate 5.

[0029] (4) Dissolve the intermediate 5 in dichloromethane in an ice-water bath, and add a mixed solution of 2M HCl-dioxane and dichloromethane dropwise; after the addition is complete, transfer to room temperature for reaction; after about 6 hours, the reaction solution is concentrated under reduced pressure to obtain a light red oil; after adding ethyl acetate, a large amount of white solid precipitates; filter, wash the obtained solid with ethyl acetate / petroleum ether, and dry to obtain the hydrochloride of the intermediate 6;

[0030] (5) 4-deuterated methoxynicotinic acid or 4-methylthionicotinic acid and HATU were added to dichloromethane, activated in an ice-water bath for 20 min, and then N,N,-diisopropylethylamine and the hydrochloride of intermediate 6 were added. The reaction was allowed to react at room temperature for 12 h and monitored by TLC. After the reaction was completed, water was added, and the dichloromethane phase obtained by separation was washed with a saturated aqueous sodium bicarbonate solution and a saturated sodium chloride solution in turn, dried over anhydrous sodium sulfate, filtered, and separated by a flash column to obtain the target compound.

[0031] 3. Biological activities and applications of deuterated piperazine compounds

[0032] The present invention discloses the activity screening results of deuterated piperazine compounds and their first application as main protease inhibitors. Experiments prove that the deuterated piperazine compounds of the present invention can be used as main protease inhibitors for preparing anti-coronavirus drugs. The present invention also provides the application of the above compounds in preparing anti-coronavirus drugs.

[0033] Anti-SARS-CoV-2 main protease activity test of target compounds

[0034] The four deuterated piperazine compounds synthesized according to the above method were tested for their activity against the SARS-CoV-2 main protease target, and their anti-main protease activity data are listed in the attached Figure 2 In the experiment, the lead compound GC-78 and the marketed drug namatevir were used as positive controls.

[0035] The newly synthesized deuterated piperazine compounds of the present invention all exhibit excellent main protease inhibitory activity. Among them, the activity of GC-78d is particularly outstanding (IC 50 =0.21 μM). The antiviral activity screening results on Vero E6 cells showed that GC-78d had the best activity (EC 50 =0.25 μM), compared with the lead compound GC-78 (EC 50 =0.89 μM) was increased by 3 times. Therefore, deuterated piperazine compounds can be used as anti-SARS-CoV-2 lead compounds for further research and development.

[0036] A class of deuterated piperazine compounds of the present invention can be used as SARS-CoV-2 main protease inhibitors, specifically, as SARS-CoV-2 inhibitors for the preparation of anti-new coronavirus drugs.

[0037] An anti-new coronavirus pharmaceutical composition comprises a deuterated piperazine compound of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0038] The present invention provides a deuterated piperazine compound with a novel structure and a preparation method thereof. The present invention also provides the results of screening the activity of the compound against the main protease of SARS-CoV-2 and its first application in the field of antiviral drugs. The deuterated piperazine compound of the present invention can be used as a SARS-CoV-2 main protease inhibitor and has a high application value. Specifically, the present invention has discovered a main protease inhibitor with higher activity and novel structure through structural optimization, which can be used as a SARS-CoV-2 main protease inhibitor for the preparation of anti-new coronavirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is M pro Three-dimensional structure and active center diagram;

[0040] Figure 2 SARS-CoV-2M pro Schematic diagram of drug screening for targets. DETAILED DESCRIPTION

[0041] The following examples are helpful for understanding the present invention, but they cannot limit the content of the present invention. The percentages are all mass percentages.

[0042] Example 1: Preparation of representative compounds

[0043]

[0044] (1) Add (S)-1-tert-butyl-3-methylpiperazine-1,3-dicarboxylic acid (1, 5.0 g, 20.5 mmol, 1.0 eq.) and 3,4-dichlorophenylboronic acid (2, 7.8 g, 41 mmol, 2.0 eq.) to 100 mL of dichloromethane and dissolve until almost clear during stirring. Add anhydrous copper acetate (3.71 g, 20.5 mmol, 1.0 eq.) and pyridine (3.20 g, 41 mmol, 2.0 eq.) to the solution at once. After the suspension is uniform, react under oxygen atmosphere for 24 h. TLC detection of the reaction EA / PE = 1:2, v / v. After 16 h of complete reaction, add water to the system to quench the reaction. Separate the liquid and wash repeatedly with distilled water until the aqueous phase has no blue color. The organic phase was washed with a saturated sodium chloride solution, separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by a Flash column (EA / PE gradient elution) to obtain intermediate 3, which was a colorless oily liquid (4.5 g) with a yield of 59%. 17 H 22 Cl2N2O4(388.1). 1 HNMR (400MHz, DMSO-d6) δ7.41(d,J=9.0Hz,1H),7.13(s,1H),6.83(dd,J=47.5,8.8Hz,1H),4.91–4.70(m,1H),4.38(d,J=13. 5Hz,1H),3.96(t,J=16.6Hz,1H),3.60(s,3H),3.50(d,J=11.5Hz,1H),3.24–2.89(m,2H),1.40(d,J=1.6Hz,9H).ESI-MS:m / z 389.2[M+H] + .C 17 H 22 Cl2N2O4(388.1).

[0045] (2) Intermediate 3 (4.0 g, 7.5 mmol, 1.0 eq.) was dissolved in a mixture of 50 mL of methanol and 50 mL of tetrahydrofuran, and an aqueous solution of lithium hydroxide (1.57 g, 37.5 mmol, 5.0 eq.) was added dropwise under ice bath. The mixture was reacted at room temperature. After stirring for 12 h, part of the organic solvent was concentrated under reduced pressure. 1 M dilute hydrochloric acid solution was added dropwise to the remaining aqueous solution under ice bath to adjust the pH to 4. A large amount of milky white solid precipitated. The mixture was filtered, washed with 100 mL of water for several times, and dried under vacuum to obtain about 2.5 g of intermediate 4, with a yield of 89.3%. It was a slightly yellow powdery solid. ESI-MS: m / z 373.1 [MH] - .C 16 H 20 Cl2N2O4(374.0).

[0046] (3) Under ice-water bath, intermediate 4 (2.4 g, 6.4 mmol, 1.0 eq.) and HATU (3.65 g, 9.6 mmol, 1.5 eq.) were added to 100 mL of dichloromethane. After activation for 30 min, diisopropylethylamine (DIPEA, 2.5 g, 19.2 mmol, 3.0 eq.) and deuterated methoxyaniline (1.2 g, 9.7 mmol, 1.0 eq.) were added. The mixture was allowed to react at room temperature for 20 hours. The reaction was completed after TLC monitoring. Water was added to quench the reaction. After extraction with dichloromethane, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by silica gel column chromatography to obtain 2.4 g of the target product with a yield of 79.1%. ESI-MS: m / z 483.2 [M+H] + .C 21 H 25 Cl2N3O3S(482.1).

[0047] (4) Dissolve the intermediate 5 (2.45 g, 5.1 mmol, 1.0 eq.) in 100 mL of dichloromethane under an ice-water bath, and add a mixed solution of 6 mL of 2M HCl-dioxane and 30 mL of dichloromethane dropwise. After the addition is complete, the mixture is allowed to react at room temperature. After about 5 h, the reaction is complete as determined by TLC, and the reaction solution is concentrated under reduced pressure to obtain a light red oil. After adding 50 mL of ethyl acetate, a large amount of white solid is precipitated. Filter, wash the solid twice with 30 mL of ethyl acetate, and dry in vacuo. 1.7 g of the hydrochloride salt of the target product intermediate 6 is obtained, with a yield of 73.5%. ESI-MS: m / z 383.1 [M+H] + .C 16 H 17 Cl2N3OS(382.1).

[0048] (5) 4-deuterated methoxynicotinic acid (0.037 g, 0.24 mmol, 1.2 eq.) and HATU (0.114 g, 0.3 mmol, 1.5 eq.) were added to 10 mL of dichloromethane, activated for 30 min, and then diisopropylethylamine (DIPEA, 0.103 g, 0.8 mmol, 4.0 eq.) and the hydrochloride of the key intermediate 6 (0.091 g, 0.2 mmol, 1.0 eq.) were added. The reaction was allowed to react at room temperature for 12 h and monitored by TLC. After the reaction was completed, water was added to quench the reaction, and the dichloromethane phase was separated and washed with a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and separated by silica gel column chromatography to obtain the target compound GC-78d. The product was a white powder with a yield of 58% and a melting point of 138-139°C.

[0049] 1 HNMR(600MHz,DMSO-d6)δ10.09(s,1H),9.73(s,1H),8.58–8.18(m,2H),7.42(d,J=10.3Hz,2H),7.31(s,1H),7.16(s,1H),7.05(s,1H),6.92–6. 77(m,3H),4.33(s,1H),3.81–3.72(m,1H),3.67(dd,J=8.3,4.1Hz,1H), 3.63–3.57(m,1H),3.54(dd,J=13.6,4.7Hz,1H),3.45(d,J=11.8Hz,1H). 13 C NMR (150MHz, DMSO) δ168.72,165.11,161.40,156.00,152.66,152.46,150.14,132.02,131.04,121.99,121.88,115 .77,115.59,114.65,114.50,114.30,114.18(C×2),107.78,58.73,54.04,45.58,43.29,41.26,40.52.ESI-MS:m / z 521.54[M+H] + .C 25 H 18 D6Cl2N4O4(520.1).HPLC purity:99.43%.

[0050]

[0051] The operation is the same as above, except that p-methoxyaniline is used in step 3.

[0052] The product is a light yellow foamy solid, yield: 45%, melting point: 115-116°C.

[0053] 1 HNMR(600MHz,DMSO-d6)δ10.09(s,1H),9.73(s,1H),8.60–7.90(m,2H),7.42 (d,J=10.3Hz,2H),7.31(s,1H),7.16(s,1H),7.05(s,1H),6.87(dd,J=19.3,9 .1Hz,3H),4.33(s,1H),3.75(d,J=4.7Hz,1H),3.72(s,3H),3.68(q,J=4.4Hz, 1H), 3.63–3.56 (m, 1H), 3.54 (dd, J=13.6, 4.7Hz, 1H), 3.45 (d, J=12.2Hz, 1H). 13 C NMR (150MHz, DMSO) δ165.23,165.11,161.42,156.00,152.66,152.47,149.95,132.01,131.04,121.99,121.88,115 .79,115.59,114.66,114.50,114.31,114.19(C×2),107.96,55.66,54.03,45.58,43.29,41.26,40.51.ESI-MS:m / z 518.18[M+H] + .C 25 H 21 D3Cl2N4O4(517.1).HPLC purity:98.82%.

[0054]

[0055] The procedure is the same as above, except that 4-methoxynicotinic acid is used in step 5.

[0056] The product is a white foamy solid, yield: 67% 118-119°C.

[0057] 1HNMR(600MHz,DMSO-d6)δ10.09(s,1H),9.73(s,1H),8.55–8.40(m,1H),8.26(d,J=37.9Hz, 1H),7.42(dd,J=9.1,1.5Hz,2H),7.31(s,1H),7.16(s,1H),7.05(s,1H),6.93–6.78(m,3H) ,4.62–4.55(m,1H),4.33(s,1H),3.87(d,J=12.1Hz,1H),3.74(dd,J=13.8,4.4Hz,1H),3.6 7(dd,J=8.5,4.2Hz,1H),3.61(s,3H),3.54(dd,J=13.7,4.7Hz,1H),3.45(d,J=11.8Hz,1H). 13 C NMR (150MHz, DMSO) δ165.23,165.11,161.42,156.00,152.67,152.48,149.96,132.02,131.04,121.99,121.89,115 .77,115.59,114.66,114.50,114.30,114.18(C×2),107.79,54.00,45.58,43.28,42.26,41.26,40.50.ESI-MS:m / z 518.26[M+H] + .C 25 H 21 D3Cl2N4O4(517.1).HPLC purity:99.52%.

[0058]

[0059] The operation is the same as above, except that 4-methylthionicotinic acid is used in step 5.

[0060] The product is a light yellow solid, yield: 51%, 124-126°C.

[0061] 1HNMR(600MHz,DMSO-d6)δ9.74(s,1H),8.44(dd,J=48.6,5.0Hz,1H),8.31–8.02(m,1H),7.42(d,J=9.1Hz,2H),7.34(d,J=9.2Hz,2H),7.06(d,J=3.0Hz ,1H),6.92–6.78(m,3H),4.87–4.56(m,1H),4.35(d,J=15.9Hz,1H),3.80(s ,1H),3.73–3.54(m,2H),3.48(t,J=19.1Hz,1H),3.30(s,1H),2.47(s,3H). 13 C NMR (150MHz, DMSO) δ195.95,168.37,165.85,156.00,152.73,152.50,150.04,146.48,132.03,131.05(C×2),121.93,121.66 ,119.41,116.44,115.81,114.56,114.27,114.18(C×2),58.55,55.38,45.60,43.32,40.52,13.63.ESI-MS:m / z534.29[M+H] + .C 25 H 21 D3Cl2N4O3S(533.1).HPLC purity:94.9%.

[0062] Example 3: The main protease (M) of the target compound pro ) inhibition experiment

[0063] Experimental principle:

[0064] The fluorescence resonance energy transfer (FRET) method used is a substrate structure of MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2, where MCA is a fluorescence donor and Dnp is a fluorescence acceptor or fluorescence quencher. The complete sequence contains both a fluorescent group and a fluorescent quencher. Since the two groups are close in space, the inhibitory effect of the quencher prevents the fluorescent group from producing fluorescence. pro After that, because the main protease can cut between the amino acid residues Q and S, the fluorescent group is away from the quenching group, and the fluorescence with a wavelength of 405nm is emitted under the excitation light of 320nm. M is detected by measuring the fluorescence. pro activity, which indirectly reflects the inhibitory activity of the compound (see Appendix Figure 2 )(Dai Wenhao, et al., Science. 368(6497):1331-1335, 2020. Qiao Jingxin, et al., Science. 371(6536):1374-1378, 2021.).

[0065] Experimental methods:

[0066] The inhibitory activity of the target compound on the main protease was tested using fluorescence resonance energy transfer. MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 was used as the reaction substrate. 1.5 μM SARS-CoV-2 M pro , 500μM substrate and 10μM compound were added to a 96-well plate for initial screening, incubated at 37°C for 10 minutes, and the fluorescence intensity of each group was detected using a multifunctional microplate reader. The excitation wavelength was 320nm, the emission wavelength was 405nm, and the fluorescence intensity was measured every 10s for 10min. First, the fluorescence intensity value was converted into the increase in fluorescence intensity per unit time according to the standard curve. The data of the first minute was used to obtain the rate. The change in the initial reaction rate was used to indicate the degree of inhibition of the inhibitor on the enzyme activity. The purpose of studying the inhibitory effect of the enzyme was to use the initial reaction rate of the blank control as V0, and the initial reaction rate after adding the inhibitor as V i , then the degree of inhibition of enzyme activity can be expressed as follows: i% = (1-V i ) / V0×100%. The experiment was divided into a blank control group, a positive control group and an experimental group. Compound GC-78 and namatevir were used as the positive control group of the experiment, and the compounds whose inhibition rate exceeded the positive control at a concentration of 10 μM were rescreened.

[0067] Rescreening: Select 1.5μM SARS-CoV-2M pro , 500 μM substrate and four concentration gradients (0.1 μM, 0.5 μM, 1 μM, 5 μM) were used to test the IC of the compound. 50 Each group was set up with 3 replicate wells and incubated at 37°C for 10 minutes. The fluorescence intensity of each group was detected using a multifunctional microplate reader. The excitation wavelength was 320 nm and the emission wavelength was 405 nm. The fluorescence intensity was measured every 10 seconds for 10 minutes. Finally, IC was calculated using GraphPad Prism 8 according to the inhibition rate at different concentrations. 50 The experimental results are shown in Table 1.

[0068] Table 1. Results of rescreening of deuterated piperazine compounds for inhibition of SARS-CoV-2 main protease

[0069]

[0070]

[0071] a IC 50 (μM): The concentration of compound required to inhibit the enzyme by 50%, i.e. half inhibitory concentration.

[0072] Experimental conclusion analysis:

[0073] The newly synthesized deuterated piperazine compounds of the present invention all exhibit excellent main protease inhibitory activity. The activities of compounds GC-78d, GC-78d-S1, GC-78d-S4 and GC-87 are comparable to the enzyme inhibitory activity of the lead compound GC-78, among which the activity of GC-78d is particularly outstanding (IC 50 =0.33±0.007μM); therefore, deuterated piperazine compounds are worthy of further research and development.

[0074] Example 4 Evaluation of anti-SARS-CoV-2 activity at the cellular level - immunofluorescence method

[0075] Experimental Materials:

[0076] SARS-CoV-2 virus strain; Calu-3 cells; test compound; PBS buffer; EMEM 10% fetal bovine serum FBS; anti-spike protein (S) antibody; goat anti-mouse IgG; positive control: namatevir, GC-78;

[0077] Experimental methods:

[0078] Calu-3 cells were seeded in 96-well plates at a density of 1 × 10 5 Cell wells. After the test compound (50 μL / well) was treated with increasing concentrations, the cells were infected with an equal volume of SARS-CoV-2 (final infection multiplicity MOI of 0.01) and cultured at 37°C in a biosafety level 3 laboratory for 2 days. The cells were fixed and permeabilized with acetone / methanol (1:3) solution for 10 minutes at room temperature. The viral S protein was detected using anti-spike protein S antibody (GTX632604; Genetex, USA) and AlexaFluor488-conjugated goat anti-mouse IgG antibody (Invitrogen, USA), and the cell nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen). Fluorescence images were quantitatively analyzed using the Operetta high-content screening system (PerkinElmer, USA) and the built-in Harmony software. The 50% effective concentration (EC) was calculated by determining the concentration of the chemical required to reduce the green fluorescence intensity derived from the viral S protein by 50%. 50 ).EC50 The values ​​were generated by curve fitting using Graphpad Prism 8.0. Each sample was measured at least three times in parallel and the average value and standard deviation were taken. The experimental results are shown in Table 2.

[0079] Activity results and discussion

[0080] Table 2. Results of intracellular anti-SARS-CoV-2 activity of deuterated piperazine compounds

[0081]

[0082] a EC 50 (μM): median effective concentration.

[0083] Experimental conclusion analysis:

[0084] Representative compounds with good enzyme inhibitory activity in the present invention were selected to test the anti-SARS-CoV-2 activity in Calu-3 cells. Among them, the activity of GC-78d was particularly outstanding, and its anti-SARS-CoV-2 activity was 3 times higher than that of the lead compound GC-78, and was better than the marketed drug Nematevir; therefore, deuterated piperazine compounds have the value of further research and development.

[0085] Example 5 Evaluation of pharmacokinetic activity in rats

[0086] Objective: This study aimed to observe the time course of plasma concentration of GC-78d in rats after intravenous injection and oral gavage, and to estimate the corresponding pharmacokinetic parameters and absolute bioavailability.

[0087] Methods: Six rats were divided into two groups and given GC-78d by intravenous injection and oral gavage, respectively. Blood samples of about 0.25 mL were collected from the jugular vein at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration in the intravenous injection group and at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration in the oral gavage group. The concentration of GC-78d-Hcl in rat plasma samples was determined by LC-MS / MS, and the pharmacokinetic parameters were calculated using WinNolin software.

[0088] Results: After a single intravenous injection of 2 mg / kg GC-78d and oral administration of 10 mg / kg GC-78d in rats, the main pharmacokinetic parameters of intravenous administration are shown in Table 3:

[0089] Table 3. Pharmacokinetic parameters of GC-78d in rats a

[0090]

[0091] a PK parameters (mean ± SD, n = 3); b 2 mg / kg injected; c 10 mg / kg was administered by oral gavage.

[0092] Experimental conclusion analysis:

[0093] The representative compound GC-78d with good antiviral activity in the present invention was selected for pharmacokinetic evaluation in rats. The experimental results showed that compared with the lead compound GC-78, the pharmacokinetic properties of GC-78d in rats were greatly improved, and the oral half-life was extended from 1.7h to 4.83h; the oral bioavailability was increased to 40.7%, which fully met the needs of oral preparations. Therefore, the deuterated strategy not only improved the antiviral activity, but also greatly improved the pharmacokinetic properties. Deuterated piperazine compounds are worth further research and development.

Claims

1. A deuterated piperazine compound, or a pharmaceutically acceptable salt thereof, characterized in that: It has the structure shown in general formula I: in, R1 and R2 are: methoxy, deuterated methoxy and methylthio, each independently; The compound represented by formula I is in S configuration.

2. The deuterated piperazine compound according to claim 1, characterized in that: Pharmaceutically acceptable salt forms include commonly used hydrochlorides, sulfates or phosphates as well as methanesulfonates, p-toluenesulfonates, fumarates, citrates or acetates.

3. The deuterated piperazine compound according to claim 1, characterized in that: is one of the following compounds:

4. The method for preparing deuterated piperazine compounds according to claim 1 or 2, comprising the following steps: using 3,4-dichlorophenylboronic acid 1 and (S)-Boc-piperazine carboxylate 2 as starting materials and dichloromethane as a reaction solvent, an intermediate 3 is obtained by a Chan-Lam coupling reaction under the catalysis of copper acetate; the methyl ester is then hydrolyzed with lithium hydroxide to obtain an intermediate 4; the intermediate 4 is subsequently dissolved in an appropriate amount of dichloromethane, and condensed with various R1 under the action of a condensing agent HATU to obtain an intermediate 5; the intermediate 5 is then deprotected with trifluoroacetic acid to obtain an intermediate 6; and the intermediate 6 is then subjected to an amide condensation reaction with various substituted nicotinic acids in dichloromethane under the action of HATU to obtain various target products; The synthetic route is as follows: Reagents and conditions: (i) acetic acid ketone, oxygen, pyridine, dichloromethane, room temperature; (ii) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (iii) deuterated p-methoxyaniline or p-methoxyaniline, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; (iv) 2M HCl-dioxane, dichloromethane, room temperature; (v) deuterated methoxynicotinic acid or methylthionicotinic acid, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; in, R1 and R2 are independently methoxy, deuterated methoxy and methylthio.

5. The deuterated piperazine compound according to any one of claims 1 to 3 is used in the preparation of anti-SARS-CoV-2 M pro application in medicines.

6. An anti-coronavirus pharmaceutical composition comprising the deuterated piperazine compound according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers.

7. An anti-coronavirus pharmaceutical composition as claimed in claim 6, wherein the coronavirus is specifically a new coronavirus.

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